The sequencing of nucleic acids, such as deoxyribose nucleic acid (“DNA”) includes determining the order of the nucleotide bases, (e.g., A, C, T and G), along a direction of a nucleic acid strand. The sequence provides detailed molecular level genetic information about the organism. Although many new sequencing technologies have been developed during recent years to sequence DNA more accurately, less expensively and faster than previous techniques, it is still a laborious, expensive and time consuming process to obtain sequencing information. For example, sequencing instruments using clonal amplification in drops or on slide colonies cost $300,000-600,000 and single molecule sequencing instruments cost above $750,000, which does not include the constant stream of very expensive chemicals, reagents and sample preparation protocols required. Much of the high cost of these sequencing systems is due to (a) the optical components (microscopes or wave guides) for systems which employ light detection, (b) the custom chip fabrication required for sequencing systems based on electrical detection and (c) the high cost of special labeled chemicals and reagents required in the single molecule-based systems. Widespread use of such valuable sequencing information is greatly hindered by these high costs. Accordingly, there is a great need to develop hardware and reagents that are vastly less expensive and allow the sequencing information to be obtained in a more efficient manner.
Several known sequencing techniques rely on primer extension (FIG. 1) to sequence the DNA. Primer extension includes a Primer (P) that is in solution or attached to the solid support, a Target (T) that contains the sequence to be determined, and a polymerase molecule. An example of one such primer extension-mediated technique, pyrosequencing, is shown in FIG. 2A In analogy to commonly used hybridization nomenclature, the DNA sequence attached to the surface is called the Probe and the species captured by hybridization is called the Target. If the capture Probe is extended by a DNA polymerase, then the Probe also functions as a Primer. In some sequencing protocols, the DNA to be sequenced is attached to the solid support and the primer to be extended is supplied from the solution. Together these components form a tripartite Probe-Target-Polymerase (“PTP”) complex that can initiate primer extension. When all of the necessary auxiliary reagents are present, primer extension ensues. If the correct complementary deoxynucleotide triphosphate (“dNTP”) is present, as shown in FIG. 2B, the dNTP will be incorporated into the growing primer strand (FIG. 1).
During pyrosequencing, as the PTP complex is undergoing primer extension various chemical species are released into the surrounding solution (FIG. 2A, top) including pyrophosphate (P2O74−) molecules from the cleavage of the triphosphate moiety associated with the dNTP molecules during strand incorporation. By treating the released pyrophosphate ion with a pyrophosphatase enzyme, additional chemical energy can be obtained from this hydrolysis to drive various subsequent chemical reactions. In one case, the pyrophosphate ions are coupled through various chemical species to luciferin, which emits light in proportion to the number of pyrophosphate ions released during primer extension (FIG. 2A, top). Therefore, the sequence of the target DNA strand is determined by noting how much light is released upon incorporation of the proper nucleotides.
Another example of DNA sequencing involves electrochemical detection. In this type of sequencing, when the PTP complex is undergoing primer extension protons (H+) are also released. These protons may be detected using a pH meter to detect the protons released (FIG. 2A, middle and FIG. 3). While it is not difficult to detect protons electrochemically, the relatively large distance between the PTP complex and the electrodes may be up to many microns or even millimeters. This large distance between the sample and detector, as well as the diffusion and signal response rates associated with typical pH electrodes is much greater than techniques where the diffusion distances are shorter, which can lead to longer, lower analyte concentrations on the detector and more expensive analysis times.
Accordingly, there is a need in the art for a sequencing technique that utilizes a shorter diffusion distance, is easy to use, has inexpensive hardware, uses unlabeled nucleotides and inexpensive reagents and provides a more efficient high throughput screening process.
To address these limitations, disclosed herein are compositions, apparatus, and methods that include a system where the chemical sensor that detects the sequencing reaction is an integral, internal part of the surface or bead to which the nucleic acid to be sequenced is attached. As described above, all known sequencing systems have the sequencing-detecting sensor or reagents external to and physically separated from the sequencing reactions. By eliminating the optical components, external transducing sensors and highly specialized labeled reagents, a high throughput sequencing instrument may be built, using standard, commercially available components and unlabeled nucleotide reagents, that is at least 100 times less expensive than current sequencing instruments.